Antigen stimulation of mast cells through high affinity receptors for IgE (FceR1) leads to Ca2+ mobilization and, as a consequence, the release of inflammatory mediators that are responsible for allergic diseases such as asthma. It is generally believed that inositol 1,4,5-trisphoshate (IP3) is responsible for mobilizing Ca2+ from the intracellular endoplasmic reticulum (ER) stores. Recently, the applicants have shown that the amount of IP3 produced by FceR1 stimulation is not sufficient to cause Ca2+ mobilization through FceR1, suggesting that an alternative pathway might be involved. The goal of this proposal is to test the hypothesis that the sphingosine kinase (SK) pathway via the production of sphingosine 1-phosphate (S1P) plays an important role in Ca2+ mobilization through FceR1 in mast cells. In addition, the signaling mechanisms, which are upstream and downstream of FceRI-mediated mast cells. In addition, the signaling mechanisms, which are upstream and downstream of FceR1-mediated SK activation, will be investigated. In these studies, a mast cell line, RBL-2H3 cells will be used as a model system as FceR1-mediated signaling has been extensively studied in this cell line. Strategies will be 1) To determine whether S1P is produced in the ER membrane upon FceR1 stimulation and whether this production correlates with in Ca2+ mobilization from the ER because S1P, not being diffusible to the cytosol, must act close to the site where it is produced. 2) To determine the role of SK pathway in FceR1-mediated Ca2+ mobilization by overexpressing the cytosolic SK (cSK) or by inhibiting PLCg. Since the cSK has recently been purified and cloned, the role of cSK and PLCg in FceR1-mediated Ca2+ mobilization will be investigated. 3) To identify the upstream signaling events that are activated by FceR1 and mediated SK activation. As possible candidates for the upstream signals, Syk tyrosine kinase and Ca2+-dependent serine/threonine protein phosphatase calcineurin will be investigated. 4) To identify the signaling events that are dependent on SK activation. Specifically FceR1-mediated activity of PLD and MAPK and will be examined. The long-term goal of the proposal is to develop a therapeutic intervention for allergic diseases such as asthma by elucidating the underlying mechanisms of Ca2+-dependent release of inflammatory mediators.